Use of a Heterocyclic Bcl-xL Inhibitor and Related Analogs for Removing Senescent Cells in the Treatment of Eye Diseases and Other Age-Related Conditions

ABSTRACT

A library of heterocyclic compounds has been screened to identify particular compounds that have high inhibitory capacity for the Bcl family of regulatory proteins. Compounds identified as Bcl antagonists have been further screened to select pharmaceutical agents with both high potency and high specificity for eliminating senescent cells in comparison with replicative or quiescent cells of the same tissue type. Particular structures are identified in this disclosure that eliminate senescent cells with an EC50 in the nanomole range and a specificity around or above 100-fold. In accordance with this invention, heterocyclic compounds provided in this disclosure can be formulated for the treatment of a range of age-related conditions caused or mediated by senescent cells. Such conditions are exemplified by ophthalmic conditions, pulmonary conditions, and osteoarthritis.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/675,171, filed Aug. 11, 2017, which is continuation-in-part of U.S.patent application Ser. No. 15/611,589, filed Jun. 1, 2017, pending,which is a continuation of international patent applicationPCT/US2016/016894, filed Feb. 5, 2016, published as WO 2016/127135 anddesignating the United States, through which this application claims thepriority benefit of U.S. provisional patent application 62/113,227,filed Feb. 6, 2015. The aforelisted priority applications are herebyincorporated herein by reference in their entirety for all purposes.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under Grant No.K99AG45288 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The invention relates generally to the treatment of conditions that aremediated by senescent cells. In particular, it provides a family of Bclinhibitors that are effective for eliminating such cells and aresuitable for formulating as medicaments for use in clinical medicine.

BACKGROUND

Senescent cells accumulate in tissues and organs of individuals as theyage and are found at sites of age-related pathologies. Senescent cellsare believed important to inhibiting proliferation of dysfunctional ordamaged cells and particularly to constraining development of malignancy(Campisi, Curr. Opin. Genet. Dev. 21: 107-12 (2011); Campisi, TrendsCell Biol. 11:S27-31 (2001); Prieur et al, Curr. Opin. Cell Biol. 20:150-55 (2008)). Nevertheless, the presence of senescent cells in anindividual may contribute to aging and aging-related dysfunction(Campisi, Cell 120:513-22 (2005)). Senescent cells have been causallyimplicated in certain aspects of age-related decline in health and inthe pathophysiology of several age-related diseases.

Inhibitors of the Bcl-2 pro-apoptotic pathway that may be suitable forkilling cancer cells or senescent cells are presented in pre-grantpublications US 20170056421 A1 (U. Arkansas), 20170119789 (BuckInstitute), US 20160339019 (Unity Biotechnology et al.), 2016/0122758(Yeda Research and Development), WO2015/116735 (Mayo Foundation), WO2016/014625 (U. Arkansas), WO 2016/127135 (Unity Biotechnology), and inissued U.S. Pat. Nos. 8,691,184; 9,096,625; and 9,403,856 (U. Michigan).

SUMMARY OF THE INVENTION

To develop this invention, a library of heterocyclic compounds wasscreened to identify particular compounds that have high inhibitorycapacity for the Bcl family of regulatory proteins. compounds identifiedas Bcl antagonists were further screened for compounds having a highpotency and specificity for eliminating senescent cells in comparisonwith replicative cells of the same tissue type. Particular structureswere identified that eliminate senescent cells with an EC₅₀ in thenanomole range and a specificity of over 100-fold.

The effectiveness of the chosen compounds could not be predicted on thebasis of Bcl binding by itself. Some compounds having high Bcl bindingwere not senolytic at a level that was clinically useful. Thethree-tiered screening test is included in this description as part ofthe invention.

Included in this invention is a method of selectively removing senescentcells from a cell population or tissue. This is done by contacting thecell population or tissue with a compound having a chemical structurethat comprises Formula VI or Formula VII as shown below, or aphosphorylated form thereof.

-   -   wherein:        -   R₁ and R₂ are independently C₁ to C₄ alkyl;        -   R₃, R₄ and R₅ are independently —H or —CH₃;        -   R₆ and R₇ are independently alkyl or heteroalkyl, and are            optionally cyclized;        -   X₁ is —F, —Cl, —Br, or —OCH₃;        -   X₂ is —SO₂R′ or —CO₂R′, where R′ is —H, —CH₃, or —CH₂CH₃;        -   X₃ is —SO₂CF₃ or —NO₂; and        -   X₅ is —F, —Br, —Cl, —H, or —OCH₃;    -   or alternatively, wherein:        -   R₁ and R₂ are independently C₁ to C₄ alkyl;        -   R₃ and R₄ are both —H;        -   R₅ is —H or —CH₃;        -   R₆ and R₇ are independently alkyl or heteroalkyl, and are            optionally cyclized in the manner shown in Formula VII;        -   X₁ is —F or —Cl;        -   X₂ is —SO₂R′ or —CO₂R′, where R′ is —H, —CH₃, or —CH₂CH₃;        -   X₃ is —SO₂CF₃ or —NO₂; and        -   X₅ is —F or —H;

-   -   wherein:        -   R₁ and R₂ are independently C₁ to C₄ alkyl;        -   R₃ and R₄ are independently —H or —CH₃;        -   X₁ is —F, —Cl, —Br, or —OCH₃;        -   X₂ is —SO₂R′ or —CO₂R′, where R′ is —H, —CH₃, or —CH₂CH₃;        -   X₃ is —SO₂CF₃ or —NO₂;        -   X₄ is —OH or —COOH;        -   X₅ is —F —Cl or —H; and        -   n₁ and n₂ are independently 1, 2, or 3.

The cell population or tissue can be contacted with the compound invitro, or at or around a disease site in need of treatment, such asophthalmic tissue, in an osteoarthritic joint, or in pulmonary tissue.Typically, the compound is administered in an amount less than an amountthat would be effective for removing cancer cells from the cellpopulation or tissue.

Also included in this invention are methods of treating age relatedconditions such as an ophthalmic condition by administering in or aroundthe site of the disease a pharmaceutical composition that includes acompound having a chemical structure that comprises Formula VI or VII.

The invention further provides pharmaceutical compositions, and unitdoses of such compositions that contain an amount of the compound shownin Formula VI or Formula VII. The composition contains a formulation ofthe compound suitable for local administration in or around the site ofa condition mediated by senescent cells. Formulation of the compositionand the amount of the compound in the unit dose configure thecomposition to be effective in treating the site by decreasing theseverity of one or more signs or symptoms of the condition whenadministered to the site as a single dose. The composition or dose istypically packaged with information for administration or use of thecomposition for treatment of an age-related condition.

Also provided is a screening method of identifying senolytic compoundssuitable for use in treating conditions mediated by senescent cells. Themethod comprises screening the library for binding to any one or more ofthe Bcl isoforms, particularly Bcl-xL, Bcl-2, and/or Bcl-w in anycombination, and selecting compounds that have sufficient affinity forthe target regulator protein(s)—below 1, 0.5 or 0.3 nanomolar, in orderof increasing preference. The method further comprises screening thelibrary for an ability to kill senescent cells (such as senescentfibroblasts) at low concentration (high potency)—having an EC₅₀ below 1,0.1, or 0.01 micromolar, in order of increasing preference. The methodfurther comprises screening the library for high specificity orselectivity for killing senescent cells compared with non-senescentcells of the same tissue type—having a specificity index (SI) above 10,50, or 200 in order of increasing preference. The compounds ultimatelyselected have high affinity for at least one of the Bcl isoforms, highpotency for killing senescent cells, and high specificity. The steps ofthe screening method can be performed in any workable order.

Certain features of the invention are referred to in the appendedclaims. Other features are referred to in the description that follows.The features described in this disclosure can be selected for use aspart of a pharmaceutical composition, a method of use in vitro, or amethod of medical therapy in any operable combination.

DRAWINGS

FIGS. 1A, 1B, and 1C show nine particular compounds selected from alibrary on the basis of binding to Bcl-2 or Bcl-xL.

FIGS. 2A, 2B, and 2C show quantitative binding affinity of the ninecompounds to Bcl isoforms Bcl-xL, Bcl-2, and Bcl-w, respectively. Eachof the compounds for which the data is shown are identified according totheir designated BM number.

FIGS. 3A, 3B, and 3C shows how effective compounds were comparedstructurally to determine what substructures contribute to the desiredproperties of the compounds.

FIG. 4 shows binding affinity to Bcl isoforms, and the effectiveconcentration (EC₅₀) for killing senescent fibroblasts (SnCs) inculture.

DETAILED DESCRIPTION

This invention provides a family of heterocyclic Bcl-xL inhibitors andrelated analogs that are specially effective for removing senescentcells and treating age related conditions. By way of illustration, suchconditions include conditions of the eye, pulmonary conditions,osteoarthritis, and atherosclerosis. The invention also includestreatment of other age-related conditions, exemplified by but notlimited to those described in the sections that follow and in theprevious disclosures to which this application claims priority.

Selection of Compounds from a Compound Library

The compounds according to Formula VI and Formula VII shown above wereselected from a compound library as having particularly beneficialproperties (potency and selectivity) for purposes of killing senescentcells and treating senescent-related conditions.

The library from which the compounds were selected include compoundshaving the general formula as shown in Formula I.

wherein: A is absent, alkaline, alkenylene, alkynylene, arylene,cycloalkylene, heteroarylene, heterocycloalkylene, aminocarbonyl,alkoxycarbonyl, an ether linkage, a sulfonyl linkage, a carbamatelinkage, a carbonate linkage, an amide linkage, a urea linkage, or anester linkage, any of which is substituted or unsubstituted: B is alkyl,alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, heterocycloalkyl, acyl,aminocarbonyl, alkoxycarbonyl, alkoxy, cycloalkyloxy,heterocycloalkyloxy, sulfonyl, carbamate, carbonate, amide, amine, urea,or ester; any of which is substituted or unsubstituted, or H; E isalkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, heterocycloalkyl,acyl, aminocarbonyl, alkoxycarbonyl, alkoxy, cycloalkyloxy,heterocycloalkyloxy, sulfonyl, carbamate, carbonate, amide, amine, urea,or ester; any of which is substituted or unsubstituted, or H; C isabsent, alkaline, alkenylene, alkynylene, arylene, cycloalkylene,heteroarylene, heterocycloalkylene, aminocarbonyl, alkoxycarbonyl, anether linkage, a sulfonyl linkage, a carbamate linkage, a carbonatelinkage, an amide linkage, a urea linkage, or an ester linkage, any ofwhich is substituted or unsubstituted; D is absent, alkaline,alkenylene, alkynylene, arylene, cycloalkylene, heteroarylene,heterocycloalkylene, aminocarbonyl, alkoxycarbonyl, an ether linkage, asulfonyl linkage, a carbamate linkage, a carbonate linkage, an amidelinkage, a urea linkage, or an ester linkage, any of which issubstituted or unsubstituted; X and Y are independently absent, O, S,CO, SO₂, SO, PO₃H, NR′, BR′, PR′, POR′, alkaline, cycloalkylene,alkenylene, cycloalkenylene, alkynylene, arylene, heteroarylene, orheterocyclylene, any of which is substituted or unsubstituted, or anycombination thereof, or X and Y are members of a ring; each R′ isindependently alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl,heteroaryl, heterocycloalkyl, acyl, aminocarbonyl, alkoxycarbonyl,alkoxy, cycloalkyloxy, heterocycloalkyloxy, sulfonyl, cycloalkeny, or H,or a pharmaceutically-acceptable salt thereof.

The library includes compounds according to Formula III, IV or V, shownhere in a phosphorylatedform.

wherein Z is (CH₂)_(n)—N(R^(a))₂ or

and wherein Q is OH, alkoxy, O(CH₂)₁₋₃, NHR, NR′(C₁₋₃alkylene),NR′(alkaline), OC(═O)(C₁₋₃alkylene), OC(═O)(alkaline), C(═O)O(alkaline),C(═O)O(C₁₋₃alkylene), NHC(═O)(alkaline), NHC(═O)(C₁₋₃alkylene),C(═O)NHR, C(═O)NH(alkaline), C(═O)NH(C₁₋₃alkylene); R_(c) is hydrogen,CN, NO₂, CF₃, halo, hydroxyalkyl, optionally substituted alkoxy,optionally substituted alkyl, optionally substituted cycloalkyl,optionally substituted alkenyl, optionally substituted cycloalkenyl,optionally substituted alkynyl, optionally substituted aryl, optionallysubstituted heteroaryl, or optionally substituted heterocycloalkyl; n,r, and s are independently 1, 2, 3, 4, 5, or 6. X, Z, the A ring, R₁,R₂, R₃, R₄, R₅, R₆, R₇, R₈, R′, R″, R″′, R″ and R according to theseformulae are defined in priority application PCT/US2016/016894 (WO2016/127135).

Screening of Compounds for Apoptosis Factor Binding Activity andSenolysis

Compounds can be screened on a molecular level for their ability toinhibit the binding between Bcl-2 or Bcl-xL and their respective cognateligand. Example 1 below provides an illustration of a homogeneous assayfor this purpose.

Alternatively or in addition, compounds can be screened for biologicalactivity in an assay using senescent cells. Cultured cells are contactedwith the compound, and the degree of cytotoxicity or inhibition of thecells is determined. The ability of the compound to kill or inhibitsenescent cells can be compared with the effect of the compound onnormal cells that are freely dividing at low density, and normal cellsthat are in a quiescent state at high density. Example 2 provides anillustration using the human lung fibroblast IMR90 cell line.

Ophthalmic Conditions

Diseases of the eye that can be treated according to this inventioninclude presbyopia, macular degeneration (including wet or dry AMD),diabetic retinopathy, and glaucoma.

Macular degeneration is a neurodegenerative condition that can becharacterized as a back-of-the-eye disease, It causes the loss ofphotoreceptor cells in the central part of retina, called the macula.Macular degeneration can be dry or wet. The dry form is more common thanthe wet, with about 90% of age-related macular degeneration (AMD)patients diagnosed with the dry form. The wet form of the disease canlead to more serious vision loss. Age and certain genetic factors andenvironmental factors can be risk factors for developing AMD.Environmental factors include, for example, omega-3 fatty acids intake,estrogen exposure, and increased serum levels of vitamin D. Genetic riskfactors can include, for example, reduced ocular Dicer1 levels, anddecreased micro RNAs, and DICER ablation.

Dry AMD is associated with atrophy of the retinal pigment epithelium(RPE) layer, which causes loss of photoreceptor cells. The dry form ofAMD can result from aging and thinning of macular tissues and fromdeposition of pigment in the macula. With wet AMD, new blood vessels cangrow beneath the retina and leak blood and fluid. Abnormally leakychoroidal neovascularization can cause the retinal cells to die,creating blind spots in central vision. Different forms of maculardegeneration can also occur in younger patients. Non-age relatedetiology can be linked to, for example, heredity, diabetes, nutritionaldeficits, head injury, or infection.

The formation of exudates, or “drusen,” underneath the Bruch's membraneof the macula is can be a physical sign that macular degeneration candevelop. Symptoms of macular degeneration include, for example,perceived distortion of straight lines and, in some cases, the center ofvision appears more distorted than the rest of a scene; a dark, blurryarea or “white-out” appears in the center of vision; or color perceptionchanges or diminishes.

Another back-of-the-eye disease is diabetic retinopathy (DR). Accordingto Wikipedia, the first stage of DR is non-proliferative, and typicallyhas no substantial symptoms or signs. NPDR is detectable by fundusphotography, in which microaneurysms (microscopic blood-filled bulges inthe artery walls) can be seen. If there is reduced vision, fluoresceinangiography can be done to see the back of the eye. Narrowing or blockedretinal blood vessels can be seen clearly and this is called retinalischemia (lack of blood flow). Macular edema in which blood vessels leaktheir contents into the macular region can occur at any stage of NPDR.The symptoms of macular edema are blurred vision and darkened ordistorted images that are not the same in both eyes. Ten percent (10%)of diabetic patients will have vision loss related to macular edema.Optical Coherence Tomography can show the areas of retinal thickening(due to fluid accumulation) of macular edema.

In the second stage of DR, abnormal new blood vessels(neovascularisation) form at the back of the eye as part ofproliferative diabetic retinopathy (PDR); these can burst and bleed(vitreous hemorrhage) and blur the vision, because these new bloodvessels are fragile. The first time this bleeding occurs, it may not bevery severe. In most cases, it will leave just a few specks of blood, orspots floating in a person's visual field, though the spots often goaway after few hours. These spots are often followed within a few daysor weeks by a much greater leakage of blood, which blurs the vision. Inextreme cases, a person may only be able to tell light from dark in thateye. It may take the blood anywhere from a few days to months or evenyears to clear from the inside of the eye, and in some cases the bloodwill not clear. These types of large hemorrhages tend to happen morethan once, often during sleep. On funduscopic exam, a doctor will seecotton wool spots, flame hemorrhages (similar lesions are also caused bythe alpha-toxin of Clostridium novyi), and dot-blot hemorrhages.

Presbyopia is an age-related condition where the eye exhibits aprogressively diminished ability to focus on near objects as the speedand amplitude of accommodation of a normal eye decreases with advancingage. Loss of elasticity of the crystalline lens and loss ofcontractility of the ciliary muscles can cause presbyopia. Age-relatedchanges in the mechanical properties of the anterior lens capsule andposterior lens capsule suggest that the mechanical strength of theposterior lens capsule decreases significantly with age.

The laminated structure of the capsule of the eye also changes and canresult, at least in part, from a change in the composition of thetissue. The major structural component of the lens capsule is basementmembrane type IV collagen that is organized into a three-dimensionalmolecular network. Type IV collagen is composed of six homologous achains (al-6) that associate into heterotrimeric collagen IV protomerswith each comprising a specific chain combination of α112, α345, orα556. Protomers share structural similarities of a triple-helicalcollagenous domain with the triplet peptide sequence of Gly-X-Y, endingin a globular C-terminal region termed the non-collagenous 1 (NC1)domain. The N-termini are composed of a helical domain termed the 7Sdomain, which is also involved in protomer-protomer interactions.

Collagen IV can influence cellular function and tissue stabilization.Posterior capsule opacification (PCO) develops as a complication inapproximately 20-40% of patients in subsequent years after cataractsurgery. PCO results from proliferation and activity of residual lensepithelial cells along the posterior capsule in a response akin to woundhealing. Growth factors, such as fibroblast growth factor, transforminggrowth factor β, epidermal growth factor, hepatocyte growth factor,insulin-like growth factor, and interleukins IL-1 and IL-6, can alsopromote epithelial cell migration. In vitro, collagen IV can promoteadherence of lens epithelial cells. Adhesion of the collagen IV,fibronectin, and laminin to the intraocular lens can inhibit cellmigration and can reduce the risk of PCO.

Compounds provided by this disclosure can slow the disorganization ofthe type IV collagen network, decrease or inhibit epithelial cellmigration and can also delay the onset of presbyopia or decrease or slowthe progressive severity of the condition. They can also be useful forpost-cataract surgery to reduce the likelihood of occurrence of PCO.

Another condition treatable according to this invention is glaucoma.Normally, clear fluid flows into and out of the front part of the eye,known as the anterior chamber. In individuals who have open/wide-angleglaucoma, the clear fluid drains too slowly, leading to increasedpressure within the eye. If left untreated, the high pressure in the eyecan subsequently damage the optic nerve and can lead to completeblindness. The loss of peripheral vision is caused by the death ofganglion cells in the retina. The effect of a therapy on inhibitingprogression of glaucoma can be monitored by automated perimetry,gonioscopy, imaging technology, scanning laser tomography, HRT3, laserpolarimetry, GDX, ocular coherence tomography, ophthalmoscopy, andpachymeter measurements that determine central corneal thickness.

Cardiovascular Conditions and Atherosclerosis

Cardiovascular conditions that can potentially be treated according tothis invention include arrhythmia, atherosclerosis, cardiomyopathy,congestive heart failure, coronary artery disease (CAD), carotid arterydisease, endocarditis, heart attack, coronary thrombosis, myocardialinfarction [MI], high blood pressure/hypertension, aortic aneurysm,brain aneurysm, cardiac fibrosis, cardiac diastolic dysfunction,hypercholesterolemia/hyperlipidemia, mitral valve prolapse, peripheralvascular disease, peripheral artery disease (PAD), cardiac stressresistance, and stroke.

The methods for treating a cardiovascular disease that is associatedwith or caused by arteriosclerosis can reduce the likelihood ofoccurrence of high blood pressure/hypertension, angina, stroke, andheart attack, coronary thrombosis, and myocardial infarction (MI).Methods are provided by this invention for stabilizing atheroscleroticplaque(s) in a blood vessel, for example, an artery, of a subject,thereby reducing the likelihood of occurrence or delaying the occurrenceof a thrombotic event, such as stroke or MI. The active compound mayreduce the lipid content of an atherosclerotic plaque in a blood vessel,for example, an artery, of the subject, or increase the fibrous capthickness by, for example, causing an increase, enhancing or promotingthickening of the fibrous cap.

Atherosclerosis is characterized by patchy intimal plaques, atheromas,that encroach on the lumen of medium-sized and large arteries; theplaques contain lipids, inflammatory cells, smooth muscle cells, andconnective tissue. Atherosclerosis can affect large and medium-sizedarteries, including the coronary, carotid, and cerebral arteries, theaorta and branches thereof, and major arteries of the extremities.

Methods are provided in this disclosure for inhibiting, reducing, orcausing a decrease in, the formation of atherosclerotic plaques byadministering a compound of this invention to a subject in need thereof.Reduction in the amount of plaque in a blood vessel, for example, anartery, can be determined, for example, by a decrease in surface area ofthe plaque, or by a decrease in the extent, degree, or percent ofocclusion of a blood vessel, for example, an artery, which can bedetermined by angiography or other visualizing methods.

Also provided by this invention are methods for increasing, improving,promoting, or enhancing the stability of atherosclerotic plaques thatare present in one or more blood vessels, for example, one or morearteries of a subject.

Atherosclerosis can be a hardening or furring of the arteries and iscaused by the formation of multiple atheromatous plaques within thearteries. Atherosclerosis (also called arteriosclerotic vascular diseaseor ASVD) is a form of arteriosclerosis in which an artery wall thickens.Symptoms develop when growth or rupture of the plaque reduces orobstructs blood flow; and the symptoms can vary depending on whichartery is affected. Atherosclerotic plaques can be stable or unstable.Stable plaques regress, remain static, or grow slowly, sometimes overseveral decades, until they can cause stenosis or occlusion. Unstableplaques are vulnerable to spontaneous erosion, fissure, or rupture,causing acute thrombosis, occlusion, and infarction long before theycause hemodynamically significant stenosis. Clinical events can resultfrom unstable plaques, which do not appear severe on angiography; thus,plaque stabilization can be a way to reduce morbidity and mortality.Plaque rupture or erosion can lead to major cardiovascular events suchas acute coronary syndrome and stroke. Disrupted plaques can have agreater content of lipid, macrophages, and have a thinner fibrous capthan intact plaques.

Atherosclerosis is a syndrome affecting arterial blood vessels due insignificant part to a chronic inflammatory response of white blood cellsin the walls of arteries. This is promoted by low-density lipoproteins(LDL), plasma proteins that carry cholesterol and triglycerides, in theabsence of adequate removal of fats and cholesterol from macrophages byfunctional high-density lipoproteins (HDL). The earliest visible lesionof atherosclerosis is the “fatty streak,” which is an accumulation oflipid-laden foam cells in the intimal layer of the artery. The hallmarkof atherosclerosis is atherosclerotic

Diagnosis of atherosclerosis and other cardiovascular disease can bebased on symptoms, for example, angina, chest pressure, numbness orweakness in arms or legs, difficulty speaking or slurred speech,drooping muscles in face, leg pain, high blood pressure, kidney failureand/or erectile dysfunction, medical history, and/or physicalexamination of a patient. Diagnosis can be confirmed by angiography,ultrasonography, or other imaging tests. Subjects at risk of developingcardiovascular disease include those having any one or more ofpredisposing factors, such as a family history of cardiovascular diseaseand those having other risk factors, for example, predisposing factorsincluding high blood pressure, dyslipidemia, high cholesterol, diabetes,obesity and cigarette smoking, sedentary lifestyle, and hypertension.

The condition can be assessed, for example, by angiography,electrocardiography, or stress test. The effects of the treatment of acompound can be analyzed by, for example, comparing symptoms of patientssuffering from or at risk of cardiovascular disease that have receivedthe treatment with those of patients without such a treatment or withplacebo treatment.

Osteoarthritis

Osteoarthritis degenerative joint disease is characterized byfibrillation of the cartilage at sites of high mechanical stress, bonesclerosis, and thickening of the synovium and the joint capsule.Fibrillation is a local surface disorganization involving splitting ofthe superficial layers of the cartilage. The early splitting istangential with the cartilage surface, following the axes of thepredominant collagen bundles. Collagen within the cartilage becomesdisorganized, and proteoglycans are lost from the cartilage surface. Inthe absence of protective and lubricating effects of proteoglycans in ajoint, collagen fibers become susceptible to degradation, and mechanicaldestruction ensues. Predisposing risk factors for developingosteoarthritis include increasing age, obesity, previous joint injury,overuse of the joint, weak thigh muscles, and genetics. Symptoms ofosteoarthritis include sore or stiff joints, particularly the hips,knees, and lower back, after inactivity or overuse; stiffness afterresting that goes away after movement; and pain that is worse afteractivity or toward the end of the day.

Compounds according to this invention can be used to reduce or inhibitloss or erosion of proteoglycan layers in a joint, reduces inflammationin the affected joint, and promotes, stimulates, enhances, or inducesproduction of collagen, for example, type 2 collagen. The compound maycauses a reduction in the amount, or level, of inflammatory cytokines,such as IL-6, produced in a joint and inflammation is reduced. Thecompounds can be used for treating osteoarthritis and/or inducingcollagen, for example, Type 2 collagen, production in the joint of asubject. A compound also can be used for decreasing, inhibiting, orreducing production of metalloproteinase 13 (MMP-13), which degradescollagen in a joint, and for restoring proteoglycan layer or inhibitingloss and/or degradation of the proteoglycan layer. Treatment with acompound thereby may also reduce the likelihood of, inhibits, ordecreases erosion, or slows erosion of the bone. The compound may beadministered directly to an osteoarthritic joint, for example,intra-articularly, topically, transdermally, intradermally, orsubcutaneously. The compound may also restore, improve, or inhibitdeterioration of strength of a join, and reduce joint pain.

Pulmonary Conditions

Pulmonary conditions that can be treated according to this inventioninclude idiopathic pulmonary fibrosis (IPF), chronic obstructivepulmonary disease (COPD), asthma, cystic fibrosis, bronchiectasis, andemphysema.

COPD is a lung disease defined by persistently poor airflow resultingfrom the breakdown of lung tissue, emphysema, and the dysfunction of thesmall airways, obstructive bronchiolitis. Primary symptoms of COPDinclude shortness of breath, wheezing, chest tightness, chronic cough,and excess sputum production. Elastase from cigarette smoke-activatedneutrophils and macrophages can disintegrate the extracellular matrix ofalveolar structures, resulting in enlarged air spaces and loss ofrespiratory capacity. COPD can be caused by, for example, tobacco smoke,cigarette smoke, cigar smoke, secondhand smoke, pipe smoke, occupationalexposure, exposure to dust, smoke, fumes, and pollution, occurring overdecades thereby implicating aging as a risk factor for developing COPD.

The processes that cause lung damage include, for example, oxidativestress produced by the high concentrations of free radicals in tobaccosmoke, cytokine release due to the inflammatory response to irritants inthe airway, and impairment of anti-protease enzymes by tobacco smoke andfree radicals, allowing proteases to damage the lungs. Geneticsusceptibility can also contribute to the disease. In about 1% percentof people with COPD, the disease results from a genetic disorder thatcauses low level production of alpha-1-antitrypsin in the liver.Alpha-1-antitrypsin is normally secreted into the bloodstream to helpprotect the lungs.

Pulmonary fibrosis is a chronic and progressive lung diseasecharacterized by stiffening and scarring of the lung, which can lead torespiratory failure, lung cancer, and heart failure. Fibrosis isassociated with repair of epithelium. Fibroblasts are activated,production of extracellular matrix proteins is increased, andtransdifferentiation to contractile myofibroblasts contribute to woundcontraction. A provisional matrix plugs the injured epithelium andprovides a scaffold for epithelial cell migration, involving anepithelial-mesenchymal transition (EMT). Blood loss associated withepithelial injury induces platelet activation, production of growthfactors, and an acute inflammatory response. Normally, the epithelialbarrier heals and the inflammatory response resolves. However, infibrotic disease the fibroblast response continues, resulting inunresolved wound healing. Formation of fibroblastic foci is a feature ofthe disease, reflecting locations of ongoing fibrogenesis.

Subjects at risk of developing pulmonary fibrosis include, for example,those exposed to environmental or occupational pollutants, such asasbestosis and silicosis; those who smoke cigarettes; those who have aconnective tissue diseases such as RA, SLE, scleroderma, sarcoidosis, orWegener's granulomatosis; those who have infections; those who takecertain medications, including, for example, amiodarone, bleomycin,busufan, methotrexate, and nitrofurantoin; those subject to radiationtherapy to the chest; and those whose family member have pulmonaryfibrosis.

Symptoms of COPD can include any one of shortness of breath, wheezing,chest tightness, having to clear one's throat first thing in the morningbecause of excess mucus in the lungs, a chronic cough that producessputum that can be clear, white, yellow or greenish, cyanosis, frequentrespiratory infections, lack of energy, and unintended weight loss.Subjects with COPD can also experience exacerbations, during whichsymptoms worsen and persist for days or longer. Symptoms of pulmonaryfibrosis include, for example, shortness of breath, particularly duringexercise; dry, hacking cough; fast, shallow breathing; gradual,unintended weight loss; fatigue; aching joints and muscles; and clubbingof the fingers or toes.

Other pulmonary conditions that can be treated by using a compoundaccording to this condition include emphysema, asthma, bronchiectasis,and cystic fibrosis. Pulmonary diseases can also be exacerbated bytobacco smoke, occupational exposure to dust, smoke, or fumes,infection, or pollutants that contribute to inflammation.

Bronchiectasis can result from damage to the airways that causes them towiden and become flabby and scarred. Bronchiectasis can be caused by amedical condition that injures the airway walls or inhibits the airwaysfrom clearing mucus. Examples of such conditions include cystic fibrosisand primary ciliary dyskinesia (PCD). When only one part of the lung isaffected, the disorder can be caused by a blockage rather than a medicalcondition.

The methods of this invention for treating or reducing the likelihood ofa pulmonary condition can also be used for treating a subject who isaging and has loss of pulmonary function, or degeneration of pulmonarytissue. The respiratory system can undergo various anatomical,physiological and immunological changes with age. The structural changesinclude chest wall and thoracic spine deformities that can impair thetotal respiratory system compliance resulting in increased effort tobreathe. The respiratory system undergoes structural, physiological, andimmunological changes with age. An increased proportion of neutrophilsand lower percentage of macrophages can be found in bronchoalveolarlavage (BAL) of older adults compared with younger adults. Persistentlow grade inflammation in the lower respiratory tract can causeproteolytic and oxidant-mediated injury to the lung matrix resulting inloss of alveolar unit and impaired gas exchange across the alveolarmembrane seen with aging. Sustained inflammation of the lowerrespiratory tract can predispose older adults to increasedsusceptibility to toxic environmental exposure and accelerated lungfunction decline. Oxidative stress exacerbates inflammation duringaging. Alterations in redox balance and increased oxidative stressduring aging precipitate the expression of cytokines, chemokines, andadhesion molecules, and enzymes. Constitutive activation and recruitmentof macrophages, T cells, and mast cells foster release of proteasesleading to extracellular matrix degradation, cell death, remodeling, andother events that can cause tissue and organ damage during chronicinflammation.

The effects of the treatment can be determined using techniques thatevaluate mechanical functioning of the lung, for example, techniquesthat measure lung capacitance, elastance, and airway hypersensitivitycan be performed. To determine lung function and to monitor lungfunction throughout treatment, any one of numerous measurements can beobtained, for example, expiratory reserve volume (ERV), forced vitalcapacity (FVC), forced expiratory volume (FEV) (e.g., FEV in one second,FEV1), FEV1/FEV ratio, forced expiratory flow 25% to 75%, and maximumvoluntary ventilation (MVV), peak expiratory flow (PEF), slow vitalcapacity (SVC). Total lung volumes include total lung capacity (TLC),vital capacity (VC), residual volume (RV), and functional residualcapacity (FRC). Gas exchange across alveolar capillary membrane can bemeasured using diffusion capacity for carbon monoxide (DLCO). Peripheralcapillary oxygen saturation (SpO₂) can also be measured; normal oxygenlevels are typically between 95% and 100%. An SpO₂ level below 90%suggests the subject has hypoxemia. Values below 80% are consideredcritical and require intervention to maintain brain and cardiac functionand avoid cardiac or respiratory arrest.

Pharmaceutical Formulations and their Use

A pharmaceutical composition of according to this invention typicallyincludes a pharmaceutically active agent described above, in combinationwith other components, such as carriers, stabilizers, diluents,dispersing agents, suspending agents, thickening agents, and/orexcipients. Physiologically-acceptable carriers can be chosen tofacilitate uptake or duration of action at or around the treatment site.The formulation can be adapted depending upon the route ofadministration chosen. In preparing pharmaceutical compositions, thereader may refer to Remington: The Science and Practice of Pharmacy,Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995), and othersuitable reference materials.

Pharmaceutical compositions can be administered intherapeutically-effective amounts as pharmaceutical compositions eitherby systemic or by local administration, including, for example,intravenous, subcutaneous, intramuscular, oral, rectal, parenteral,ophthalmic, pulmonary, transdermal, vaginal, intravitreal, nasal, andtopical administration.

Compositions of the invention can be packaged as a kit, includingwritten instructions on the administration/use of the composition forparticular conditions. Pharmaceutical compositions can be packaged asunit dosage forms suitable for single administration of precise dosages.

Use of Bcl inhibitors according to this invention for killing senescentcells or treating senescent-related conditions typically requires adose, a frequency of administration, and an overall burden that isconsiderably less (100-fold or better) less than what is required totreat a cancer or hyperproliferative condition at or around the samesite or in the same tissue type. For example, for treatment of senescentrelated conditions, the compound is administered as a single dose or ina plurality of treatment cycles, wherein each treatment cycleindependently comprises a treatment course of from one day to 3 monthsfollowed by a non-treatment interval of at least 2 weeks.

Definitions

A “senescent cell” is generally thought to be derived from a cell typethat typically replicates, but as a result of aging or other event thatcauses a change in cell state, can no longer replicate. It remainsmetabolically active and commonly adopts a senescence associatedsecretory phenotype (SASP). The nucleus of senescent cells is oftencharacterized by senescence-associated heterochromatin foci and DNAsegments with chromatin alterations reinforcing senescence. Withoutimplying any limitation on the practice of what is claimed in thisdisclosure that is not explicitly stated or required, the invention ispremised on the hypothesis that senescent cells cause or mediate certainconditions associated with tissue damage or aging. For the purpose ofpracticing aspects of this invention, senescnet cells can be identifiedas expressing at least one marker selected from p16,senescence-associated β-galactosidase, and lipofuscin; sometimes two ormore of these markers, and optionally other markers of SASP such asinterleukin 6.

A “senescence associated” disease, disorder, or condition is aphysiological condition that presents with by one or more symptoms orsigns, wherein a subject having the condition needs or would benefitfrom a lessening of such symptoms or signs. The condition is senescenceassociated if it occurs predominantly in people over 65 years of age, orif it is caused or mediated in part by senescent cells. Lists ofsenescence associated disorder that can potentially be treated ormanaged using the methods and products taught in this disclosure includebut are not limited to those discussed in this disclosure and theprevious disclosures to which this application claims priority.

A compound is “senolytic” if it eliminates senescent cellspreferentially, compared with replicative cells of the same tissue type,or quiescent cells lacking SASP markers.

A “phosphorylated” form of a compound is a compound in which one or more—OH or —COOH groups have been substituted with a phosphate group whichis either —OPO₃H₂ or —C_(n)PO₃H (where n is 1 to 4), such that thephosphate group may be removed in vivo (for example, by enzymolysis). Anon-phosphorylated or dephosphorylated form has no such group.

Unless otherwise stated or required, all the compound structuresreferred to in the invention include conjugate acids and bases havingthe same structure, crystalline and amorphous forms of those compounds,pharmaceutically acceptable salts, and dissolved and solid formsthereof, including, for example, polymorphs, solvates, hydrates,unsolvated polymorphs (including anhydrates), conformational polymorphs,and amorphous forms of the compounds, as well as mixtures thereof.

Except where otherwise stated or required, other terms used in thespecification have their ordinary meaning.

INCORPORATION BY REFERENCE

Each and every publication and patent document cited in this disclosureis hereby incorporated herein by reference in its entirety for allpurposes to the same extent as if each such publication or document wasspecifically and individually indicated to be incorporated herein byreference.

US 2016/0339019 A1 (Laberge et al.) is hereby incorporated herein byreference in its entirety for all purposes, including the use ofsenolytic compounds to treat various age-related conditions andformulation as medicaments. U.S. Pat. Nos. 8,691,184, 9,096,625, and9,403,856 (Wang et al.) are hereby incorporated herein by reference inits entirety for all purposes, including the features of compounds inthe Bcl library, their preparation and use.

EXAMPLES Example 1: Measuring Bcl Inhibition

For senescnet cells and senescent-like cells having active Bcl activity,effective senolytic agents can be screened first by measuring binding toBcl isoforms.

This assay uses a homogenous assay technology based on oxygen channelingthat is marketed by PerkinElmer Inc., Waltham, Mass.: see Eglen et al.,Current Chemical Genomics, 2008, 1, 2-10. The test compound is combinedwith the target Bcl protein and a peptide representing the correspondingcognate ligand, labeled with biotin. The mixture is then combined withstreptavidin bearing luminescent donor beads and luminescent acceptorbeads, which proportionally reduces luminescence if the compound hasinhibited the peptide from binding to the Bcl protein.

Bcl-2, Bcl-xL and Bcl-W are available from Sigma-Aldrich Co., St. Louis,Mo. Biotinylated BIM peptide (ligand for Bcl-2) and BAD peptide (ligandfor Bcl-xL) are described in pre-grant publication US 2016/0038503 A1.AlphaScreen® Streptavidin donor beads and Anti-6×His AlphaLISA® acceptorbeads are available from PerkinElmer.

To conduct the assay, a 1:4 dilution series of the compound is preparedin DMSO, and then diluted in assay buffer. In a 96-well PCR plate, thefollowing are combined: 10 μL peptide, 10 μL test compound, and 10 μLBCL protein. The assay plate is incubated in the dark at roomtemperature for 24 h. The next day, donor beads and acceptor beads arecombined, and 5 L is added to each well. After incubating in the darkfor 30 minute, luminescence is measured using a plate reader, and theaffinity or degree of inhibition by each test compound is determined.

Example 2: Measuring Senolytic Activity

Ability of candidate agents to eliminate senescent cells orsenescent-like cells can be measured directly, for example, in thefollowing assay.

Human fibroblast IMR90 cells can be obtained from the American TypeCulture Collection (ATCC®) with the designation CCL-186. The cells aremaintained at <75% confluency in DMEM containing FBS and Pen/Strep in anatmosphere of 3% O₂, 10% CO₂, and 95% humidity. The cells are dividedinto three groups: irradiated cells (cultured for 14 days afterirradiation prior to use), proliferating normal cells (cultured at lowdensity for one day prior to use), and quiescent cells (cultured at highdensity for four day prior to use).

On day 0, the irradiated cells are prepared as follows. IMR90 cells arewashed, placed in T175 flasks at a density of 50,000 cells per mL, andirradiated at 10-15 Gy. Following irradiation, the cells are plated at100 L in 96-well plates. On days 1, 3, 6, 10, and 13, the medium in eachwell is aspirated and replaced with fresh medium.

On day 10, the quiescent healthy cells are prepared as follows. IMR90cells are washed, covered with 3 mL of TrypLE trypsin-containing reagent(Thermofisher Scientific, Waltham, Mass.) and cultured for 5 min untilthe cells have rounded up and begin to detach from the plate. Cells aredispersed, counted, and prepared in medium at a concentration of 50,000cells per mL. 100 L of the cells is plated in each well of a 96-wellplate. Medium is changed on day 13.

On day 13, the proliferating healthy cell population is prepared asfollows. Healthy IMR90 cells are washed, covered with 3 mL of TrypLE andcultured for 5 minutes until the cells have rounded up and begin todetach from the plate. Cells are dispersed, counted, and prepared inmedium at a concentration of 25,000 cells per mL. 100 L of the cells isplated in each well of a 96-well plate.

On day 14, test senolytic agents are combined with the cells as follows.A DMSO dilution series of each test compound is prepared at 200 timesthe final desired concentration in a 96-well PCR plate. Immediatelybefore use, the DMSO stocks are diluted 1:200 into prewarmed completemedium. Medium is aspirated from the cells in each well, and 100 L/wellof the compound containing medium is added.

The test senolytic agents are cultured with the cells for 6 days,replacing the culture medium with fresh medium and the same compoundconcentration on day 17. Bcl-2 inhibitors such as those tested in theexamples that follow are cultured with the cells for 3 days. The assaysystem uses the properties of a thermostable luciferase to enablereaction conditions that generate a stable luminescent signal whilesimultaneously inhibiting endogenous ATPase released during cell lysis.At the end of the culture period, 100 L of CellTiter-Glo@ reagent(Promega Corp., Madison, Wis.) is added to each of the wells. The cellplates are placed for 30 seconds on an orbital shaker, and luminescenceis measured.

Killing of virally infected senescent cells or senescent like cells canbe measured in the same manner mutatis mutandis by using senescent cellsthat are virally infected, comparing results to non-senescent and/ornon-infected cells.

Example 3: Screening a Compound Library for Bcl Antagonists

Discovery of senolytic agents useful for implementation according tothis invention was based on the premise that senescent cells can bekilled by inhibiting one or more of the Bcl family of regulator proteinsthat are anti-apoptotic. A molecule with high affinity and selectivityfor a Bcl isoform was hypothesized to be effective in inducing apoptosisin senescent cells but not proliferating or non-senescent cells of thesame tissue type. Compounds with these properties would be candidatesfor development as therapeutic agents for clinical medicine.

A library that was initially constructed that contained several hundredcompounds. Synthesis and use of such library are explained in U.S. Pat.Nos. 8,691,184, 9,096,625, and 9,403,856. The library was initiallyscreened elsewhere for compounds that were able to bind or inhibitBcl-xL and/or Bcl-2. Fifteen compounds were chosen from the initialscreening for further analysis. FIGS. 1A, 1B, and 1C show nine of thefifteen compounds.

The chosen compounds were further assayed to quantitatively determinethe actual affinity for Bcl-xL, Bcl-2, and Bcl-w with a view toidentifying candidate senolytic agents for use in treating age relatedconditions. FIGS. 2A, 2B, and 2C show the results of the binding assay,with those compounds towards the left of each graph having the highestaffinity.

Individual compounds in the library had similar core structures. Asshown in FIGS. 3A, 3B, and 3C, the structures of each of the compoundshaving the most promise were compared with a view to identifyingsubstituents of the molecular structure that were contributing to thedesired effect.

Example 4: Screening High Affinity Bcl Antagonists for Senolvtic Potencyand Specificity

An ability to bind Bcl regulatory proteins does not necessarily meanthat the compound is suitable for inducing apoptosis in a clinicalsetting. Furthermore, even if the compound is potent, it would not besuitable for use as a therapeutic unless it preferentially killssenescent cells with a high degree of selectivity. Accordingly,compounds in the library showing high Bcl binding affinity were furtherscreened for their ability to kill irradiated fibroblasts, in comparisonwith replicating fibroblasts or fibroblasts that were quiescent (due toconfluence) but not senescent.

FIG. 4 shows data obtained from the nine model compounds: affinity ofbinding to each of the Bcl isoforms, and the effective concentration(EC₅₀) for killing senescent fibroblasts (SnCs) in culture. The data aresummarized in TABLE 1, below.

Best Original Molecular Binding Affinities (K_(i) ± SD) SnCs EC₅₀ IDsWeight Bcl-xL (pM) Bcl-2 (pM) Bcl-w (pM) (μM) SI BM-501 639.1 NA NA NANA NA BM-983 967.5 3307 4037 >250000 5.000 1.0 BM-782 1274.4 885.5510923 17694 11.000 1.3 BM-788 1148.2 5292 121045 23132.5 NA NA BM-7921165.7 64.125 3324 1295.5 1.000 3.3 BM-957 1179.7 45.3 3333 659.95 0.30011.7 BM-962 1193.7 57.41 3389.5 538.15 0.200 18.5 BM-1075 1113.7 5.7285178.95 161.45 0.300 15.3 BM-1192 1215.3 NA NA NA 0.050 60.0 BM-11951229.3 148.565 666.55 174.7 0.006 128.7 BM-1197 1245.8 295 447.95 291.60.005 366.7 BM-1244 1273.8 134.52 450.75 356.75 0.300 66.7 BM-12611409.9 106.89 274.45 239.45 27.000 NA BM-1252 1395.9 81.085 135.85123.95 6.000 NA

The data show that binding to any of the Bcl isoforms with high affinitywas not necessarily predictive of an effective senolytic agent. Thecompounds designated BM-1075, BM-1195, BM-1197, BM-1244, BM-1261, andBM-1252 all had binding affinities (Ki) for the Bcl isoforms that werein the nanomole to picomole range. However, in the assay to determineeffective concentration for killing the cells (EC₅), some of thesemolecules, such as BM-1244, BM-1261, and BM-125, were 60 to 5,000-foldless potent than the compounds ultimately chosen for development.

The compounds with the best senolytic activity, BM-1195 and BM-1197,were potent in the nanomole range. There was a wide range of specificityfor senescent cells (SI) determined for the various compounds, rangingfrom 1.0 (non-specific) to over 300. The best compound in terms of bothpotency and specificity was BM-1197, with BM-1195, BM-1244, BM-1105, andBM-1075 also being of interest.

In accordance with the data, the following deductions were made withrespect to the chemical structure. This substructure was at the core ofeffective compounds:

with the R₃, R₄, and F groups being optional. The —SO₂CF₃ in BM-1197 isinfluential, but could be substituted with groups having similarproperties, such as —NO₂ in BM-1075. The —SO₂R′ group is influential,although R′ could be varied from —CH₃ to other short-chain alkyl groups.The aryl —S—C₆H₅ group is also influential, although it couldpotentially have neutral substituents. With respect to the followingpart of the structure:

R₁ can be several short-chain alkyl groups, and X₁ can be varied (Cl inBM-1197; Fin BM-1195). The following part of BM-1197:

appears to be forgiving in form, with a range of alternativesubstructures being effective for many purposes of this invention.

These and other deductions lead to the drawing of the generic structureshown earlier as Formula VI and Formula VII.

Example 5: Efficacy of Compounds in an Animal Model of Osteoarthritis

C57BL/6J mice undergo surgery to cut the anterior cruciate ligament ofone rear limb to induce osteoarthritis in the joint of that limb. Atweek 2 post-surgery, mice receive 2.5 g of test compound to the operatedknee by intra-articular injection, q.d. for 5 days, with a secondtreatment (2.5 g test compound-q.d. for 5 days) during week 4post-surgery. At the end of 4 weeks post-surgery, operated joints of themice are assessed for function, monitored for markers of inflammation,and undergo histological assessment.

Two control groups of mice are included: One group comprising C57BL/6Jmice that undergo a sham surgery (i.e., surgical procedures are followedexcept for cutting the ACL) and receive intra-articular injections ofvehicle parallel to the treated group; and one group comprising C57BL/6Jthat undergo an ACL surgery and receive intra-articular injections ofvehicle.

Function of the limbs are assessed at 4 weeks post-surgery by a weightbearing test to determine which leg the mice favor. The mice are allowedto acclimate to the chamber on at least 3 occasions prior to takingmeasurements. Mice are maneuvered inside the chamber to stand with 1hind paw on each scale. The weight that is placed on each hind limb ismeasured over a 3-second period. At least 3 separate measurements aremade for each animal at each time point. The results are expressed asthe percentage of the weight placed on the operated limb versus thecontralateral unoperated limb.

The function of the limbs are also assessed at 4 weeks post-surgery byhotplate analysis to show sensitivity and reaction to pain stimulus. Inbrief, a mouse is placed on a hotplate at 55° C. When placed on the hotsurface of the plate, mice will lift their paws and lick them (paw-lickresponse) due to attainment of pain threshold. The latency period forthe hind limb response (paw-lick response) is recorded as response time.Histopathology of the proteoglycan layer is also analyzed.

Example 6: Efficacy of Compounds in an Animal Model of Cardiac StressResistance

At 12 months of age, mice are injected three times per week with thetest compound, while a control group receives vehicle. At 18 months,subsets of male and female mice are subjected to a cardiac stress test,in which mice are injected with a lethal dose of isoproterenol (680mg/kg) and the time to cardiac arrest is recorded. The time to cardiacarrest is compared between treated and untreated animals.

Example 7: Efficacy of Compounds in an Animal Model of Cardiac StressResistance

LDL^(−/−) mice from 10 weeks of age are fed a high fat diet having 42%calories from fat beginning at Week 0 until Week 12.5. The mice are thenswitched to a normal chow diet. Mice are treated with a test compound ora vehicle from week 12.5 over the next 100 days, with each treatmentcycle comprising 5 days of test compound (25 mg/kg intraperitoneallydaily) and 14 days off. At the end of the 100 day treatment period, miceare sacrificed, plasma and tissues are collected, and atherosclerosis isquantitated. Descending aortas are dissected and stained with Sudan IVto visualize the plaque lipids. The percentage of the aorta covered inplaques is measured by area, and is compared between the treated anduntreated animals.

Example 8: Efficacy of Compounds in Animal Models of Pulmonary Disease

To assess the efficacy of compounds in treating pulmonary diseases, amodel of bleomycin-induced injury is used. In this model, mice developlung fibrosis within 7-14 days after bleomycin treatment.

Bleomycin is administered to anesthetized 6-8 week-old mice byintratracheal aspiration (2.5 U/kg of bleomycin in 50 L PBS) using amicrosprayer syringe. Control mice are administered saline. The dayfollowing bleomycin treatment, test compound (25 mg/kg in PBS) orvehicle is administered. Mice are treated via intraperitoneal injectionfor 5 consecutive days, followed by 5 days of rest, followed by a secondtreatment cycle of 5 consecutive days. Untreated mice receive an equalvolume of vehicle. At 7, 14, and 21 days post-bleomycin treatment, lungfunction is assessed by monitoring oxygen saturation using theMouseSTAT™ PhysioSuite pulse oximeter (Kent Scientific). Animals areanesthetized with isoflurane (1.5%) and a toe clip is applied. Mice aremonitored for 30 seconds and the average peripheral capillary oxygensaturation (SpO₂) measurement over this duration is calculated.

At 21 days post-bleomycin treatment, airway hyper-reactivity (AHR) ofmice is examined. AHR of mice is measured by methacholine challengewhile other parameters of lung function (airway mechanics, lung volumeand lung compliance) are determined using a SCIREQ™ flexiVentventilator. While under ketamine/xylazine anesthesia and subjected tocannulation of the trachea via a tracheostomy (19Fr blunt Luer cannula),airway resistance (elastance) and compliance of mice are assessed atbaseline and in response to increasing concentrations of methacholine (0to 50 mg/mL in PBS) delivered via nebulization (AeroNeb). Animals aremaintained at 37° C., and while under muscle paralysis (pancuronium);airway function is measured by using the FlexiVent™ ventilator and lungmechanics system (SCIREQ, Montreal, Quebec, Canada).

Mice are euthanized by intraperitoneal injection of pentobarbital.Bronchoalveolar lavage (BAL) fluids and lungs is obtained and analyzed.Hydroxyproline content of lungs is measured and quantitativehistopathology is performed.

In a second animal model for pulmonary diseases (e.g., COPD), mice areexposed to cigarette smoke. The effect of a test compound on the miceexposed to smoke is assessed by lung function and histopathology.

Six week-old mice are chronically exposed to cigarette smoke from aTeague TE-10 system, an automatically-controlled cigarette smokingmachine that produces a combination of side-stream and mainstreamcigarette smoke in a chamber, which is transported to a collecting andmixing chamber where varying amounts of air is mixed with the smokemixture. Mice receive a total of 6 hours of cigarette smoke exposure perday, 5 days a week for 6 months. Each lighted cigarette is puffed for 2seconds and once every minute for a total of 8 puffs, with the flow rateof 1.05 L/min, to provide a standard puff of 35 cm3. The smoke machineis adjusted to produce a mixture of side stream smoke (89%) andmainstream smoke (11%) by smoldering 2 cigarettes at one time. The smokechamber atmosphere is monitored for total suspended particulates (80-120mg/m³) and carbon monoxide (350 ppm). Beginning at day 7, mice aretreated with test compound or vehicle (3×per week) (5 consecutive daysof treatment followed by 16 days off drug, repeated until the end of theexperiment), respectively. An equal number of mice received thecorresponding vehicle.

After two months of cigarette smoke exposure, lung function is assessedby monitoring oxygen saturation using the MouseSTAT PhysioSuite pulseoximeter (Kent Scientific). Animals are anesthetized with isoflurane(1.5%) and the toe clip is applied. Mice are monitored for 30 secondsand the average peripheral capillary oxygen saturation (SpO₂)measurement over this duration is calculated. At the end of theexperimental period, airway hyper-reactivity (AHR) of mice tomethacholine challenge using a SCIREQ flexiVent ventilator and lungmechanics system is examined as described above. After AHR measurement,mice are killed by intraperitoneal injection of pentobarbital forin-depth analysis of lung histopathology. Briefly, lungs are inflatedwith 0.5% low-melting agarose at a constant pressure of 25 cm. Lungs arefixed in 10% buffered formalin and embedded in paraffin. Sections (5 m)are stained with hematoxylin and eosin. Mean alveolar diameter, alveolarlength, and mean linear intercepts are determined by computer-assistedmorphometry with Image Pro Plus™ software (Media Cybernetics).

The several hypotheses presented in this disclosure provide a premise byway of which the reader may understand the invention. This premise isprovided for the enrichment and appreciation of the reader. Practice ofthe invention does not require detailed understanding or application ofthe hypothesis. Except where stated otherwise, features of thehypothesis presented in this disclosure do not limit application orpractice of the claimed invention. For example, except where theelimination of senescent cells is explicitly required, the compounds ofthis invention may be used for treating the conditions describedregardless of their effect on senescent cells.

While the invention has been described with reference to the specificexamples and illustrations, changes can be made and equivalents can besubstituted to adapt to a particular context or intended use as a matterof routine development and optimization and within the purview of one ofordinary skill in the art, thereby achieving benefits of the inventionwithout departing from the scope of what is claimed.

The invention claimed is:
 1. A method of selectively removing senescentcells from a cell population or tissue, comprising contacting the cellpopulation or tissue with a compound having a chemical structure thatcomprises Formula VI as shown below or a phosphorylated form thereof.

wherein: R₁ and R₂ are independently C₁ to C₄ alkyl R₃, R₄ and R₅ areindependently —H or —CH₃; R₆ and R₇ are independently alkyl orheteroalkyl, and are optionally cyclized; X₁ is —F, —Cl, —Br, or —OCH₃;X₂ is —SO₂R′ or —CO₂R′, where R′ is —H, —CH₃, or —CH₂CH₃; X₃ is —SO₂CF₃or —NO₂; and X₅ is —F, —Br, —Cl, —H, or —OCH₃.
 2. The method of claim 1,wherein the compound has a chemical structure that comprises Formula VIas shown below or a phosphorylated form thereof.

wherein: R₁ and R₂ are independently C₁ to C₄ alkyl; R₃ and R₄ areindependently —H or —CH₃; X₁ is —F, —Cl, —Br, or —OCH₃; X₂ is —SO₂R′ or—CO₂R′, where R′ is —H, —CH₃, or —CH₂CH₃; X₃ is —SO₂CF₃ or —NO₂; X₄ is—OH or —COOH; X₅ is —F, —Cl, or —H; and n₁ and n₂ are independently 1,2, or
 3. 3. The method of claim 2, wherein the compound has one or moreof the following features in any combination: R₁ is isopropyl; R₂ ismethyl; R₃ is —H; R₄ is —H; X₁ is —Cl; X₂ is —SO₂CH₃; X₃ is —SO₂CF₃; X₄is —OH; n₁ is 2; and n₂ is
 2. 4. The method of claim 2, wherein thecompound is a phosphorylated form of Formula VII.
 5. The method of claim2, wherein the compound is a non-phosphorylated form of Formula VII. 6.The method of claim 3, wherein the compound has all of said features. 7.The method of claim 1, wherein the cell population or tissue iscontacted with the compound in vitro.
 8. The method of claim 1, whereinthe cell population or tissue is in ophthalmic tissue.
 9. The method ofclaim 1, wherein the cell population or tissue is in an osteoarthriticjoint or in pulmonary tissue.
 10. The method of claim 1, wherein thecompound is administered in an amount less than an amount that would beeffective for removing cancer cells from the cell population or tissue.11. The method of treating an ophthalmic condition mediated by senescentcells, comprising administering in or around the eye a pharmaceuticalcomposition that includes a compound having a chemical structure thatcomprises Formula VII as shown below:

wherein: R₁ and R₂ are independently C₁ to C₄ alkyl; R₃ and R₄ areindependently —H or —CH₃; X₁ is —F, —Cl, —Br, or —OCH₃; X₂ is —SO₂R′ or—CO₂R′, where R′ is —H, —CH₃, or —CH₂CH₃; X₃ is —SO₂CF₃ or —NO₂; X₄ is—OH or —COOH; X₅ is —F, —Cl, or —H; and n₁ and n₂ are independently 1,2, or
 3. 12. The method of claim 11, wherein the ophthalmic condition isa back-of-the-eye disease.
 13. The method of claim 12, wherein theophthalmic condition is age-related macular degeneration (AMD).
 14. Themethod of claim 13, wherein the ophthalmic condition is wet AMD.
 15. Themethod of claim 13, wherein the ophthalmic condition is dry AMD.
 16. Themethod of claim 11, wherein the compound is administered in a pluralityof treatment cycles, wherein each treatment cycle independentlycomprises a treatment course of from 1 day to 3 months followed by anon-treatment interval of at least 2 weeks
 17. The method of claim 11,wherein the compound is administered as a single dose followed by anon-treatment interval of at least 2 weeks.
 18. A unit dose of apharmaceutical composition for treatment of a condition in a tissuecaused or mediated by senescent cells in the tissue; wherein the unitdose contains an amount of a compound having a structure shown inFormula VI or Formula VII; wherein the composition contains aformulation of the amount that is suitable for local administration inor around a site where the senescent cells reside; and wherein theformulation of the composition and the amount of the compound in theunit dose configure the unit dose to be effective in treating thecondition at the site by decreasing the severity of one or more signs orsymptoms of the condition when administered to the site as a singledose.
 19. The unit dose according to claim 18, wherein the condition isan ophthalmic condition
 20. The unit dose according to claim 18, whereinthe condition is selected from osteoarthritis or a pulmonary condition.21. The unit dose according to claim 18, packaged with information foradministration or use of the composition for treatment of the condition.